Chemical inducible promoters used to obtain transgenic plants with a silent marker

ABSTRACT

Chemically inducible promoters are described that may be used to transform plants, including tobacco and lettuce, with genes which are easily regulatable by adding the plants or plant cells to a medium containing an inducer of the promoter or by removing the plants or plant cells from such medium. The promoters described are ones that are inducible by a glucocorticoid or estrogen which is not endogenous to plants. Such promoters may be used with a variety of genes such as ipt, CKI1, or knotted1, to induce shoot formation in the presence of an appropriate inducer. The promoters may be used with genes which induce somatic embryos such as Lec1 or SERK to prepare somatic embryos which can be grown into seedlings and then into plants. The promoter may also be used with antibiotic or herbicide resistance genes which are then regulatable by the presence or absence of inducer rather than being constitutive. Other examples of genes which may be placed under the control of the inducible promoter are also presented.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation in part of Ser. No. 09/014,592, filed Jan. 28,1998, now U.S. Pat. No. 6,063,985, which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

Transgenic techniques have become a powerful tool for addressingimportant biological problems in multicellular organisms, and this isparticularly true in the plant field. Many approaches that wereimpossible to implement by traditional genetics can now be realized bytransgenic techniques, including the introduction of homologous orheterologous genes into plants, with modified functions and alteredexpression patterns. The success of such techniques often depends uponthe use of markers to identify the transgenic plants and promoters tocontrol the expression of the transgenes.

Selectable markers are widely used in plant transformation. Historicallysuch markers have often been dominant genes encoding either antibioticor herbicide resistance (Yoder and Goldsbrough, 1994). Although suchmarkers are highly useful, they do have some drawbacks. The antibioticsand herbicides used to select for the transformed cells generally havenegative effects on proliferation and differentiation and may retarddifferentiation of adventitious shoots during the transformation process(Ebinuma et al., 1997). Also, some plant species are insensitive to ortolerant of these selective agents, and therefore, it is difficult toseparate the transformed and untransformed cells or tissues (Ebinuma etal., 1997). Further, these genes are constitutively expressed, and thereare environmental and health concerns over inserting such constitutivelyexpressed genes in plants which are grown outside of a laboratorysetting (Bryant and Leather, 1992; Gressel, 1992; Flavell et al., 1992).

One marker that is neither an antibiotic nor a herbicide is the ipt genefrom the Ti-plasmid of Agrobacterium tumefaciens. This gene encodesisopentenyltransferase, which is used in cytokinin synthesis (Barry etal., 1984). Isopentenyltransferase uses 5′-AMP and isopentenyldiphosphate to catalyze the formation ofisopentenyl-adenosine-5′-monophosphate, the first intermediate incytokinin biosynthesis. Overexpression of the ipt gene leads to elevatedcytokinin levels (Medford et al., 1989; McKenzie et al., 1998; Faiss etal., 1997; Redig et al., 1996; Ebinuma et al., 1997). Cytokinins areplant hormones that play an important role in plant development bymediating a range of morphological changes (Mok and Mok, 1994; Davies,1995; Coenen and Lomax, 1997). For example, cytokinins are able tostimulate leaf expansion and delay leaf senescence (Kuraish and Okumura,1956; Wingler et al., 1998; Gan and Amasino, 1995). In young, dark-grownseedlings, high cytokinin levels can produce a deetiolated phenotype,resembling the morphology of light-grown seedlings with shorthypocotyls, open hooks and expanded cotyledons (Chaudhury et al., 1993;Miklashevichs and Walden, 1997). Cytokinins can also release lateralbuds from apical dominance, and stimulate de novo bud formation (Cline,1991; Skoog and Miller, 1957; Sachs and Thimmann, 1967). This class ofhormones thus plays a critical role in the formation of adventitiousshoots. As demonstrated by Skoog and Miller (1957), high cytokininlevels can induce shoot differentiation from tobacco calli, aprerequisite for the regeneration of transgenic plants. Besidessupporting tumor growth, T-DNA introduction into a plant cell can alsoinduce regeneration of physiologically abnormal shoots from transformedprotoplasts or leaf disks.

Overexpression of the ipt gene (Akiyoshi et al., 1984; Barry et al.,1984), a component of the T-DNA, leads to increased cytokinin relativeto auxin, which triggers shoot regeneration (Tran Thanh Van, 1981). Thisoverproduction -of shoots can result in a phenotype of a large number ofshoots (hereafter “shooty phenotype”). This phenotype can be used as amarker (Ebinuma et al., 1997). Studies using the ipt gene under thecontrol of constitutive promoters showed that ipt overexpression causeselevated cytokinin levels in transgenic plants (Smigocki and Owens,1988; Medford et al., 1989). A chimeric ipt gene under the control ofthe cauliflower mosaic virus (CaMV) promoter has been introduced intocells of potato (Ooms et al., 1983), cucumber (Smigocki and Owens,1989), and several Nicotiana species (Smigocki and Owens, 1988) andthese transgenic cells proliferated and exhibited an extreme shootyphenotype and loss of apical dominance in hormone-free medium. Studieshave shown that in plants transformed with ipt to overproducecytokinins, the cytokinins work only locally as a paracrine hormone(Faiss et al., 1997). Grafting experiments performed with wild typetobacco plants and tobacco plants in which the ipt gene wasoverexpressed showed that the increased cytokinin levels remainedrestricted to the part of the plant that overexpressed ipt (Faiss etal., 1997).

One problem with the use of constitutively expressed ipt as a marker isthat the resulting transgenic plants lose apical dominance and areunable to root due to overproduction of cytokinins (Ebinuma et al.,1997). In addition, plants which constitutively overexpress ipt possessan altered leaf morphology and delayed leaf senescence. Such plants showlittle root growth and poor internode elongation, display delayed leafsenescence, and are very often sterile (Mok and Mok, 1994; Klee et al.,1987; Ebinuma et al., 1997).

Ebinuma et al. (1997) developed one method to use the ipt marker toovercome the problems associated with constitutive overexpression ofipt. They developed a vector in which the ipt gene was inserted into aplasmid which included the transposable element Ac. The constructincluded the T-DNA (portion of the Ti plasmid that is transferred toplant cells) and the 35S CaMV promoter. This construct was transformedinto A. tumefaciens. Leaf segments were inoculated with the transformedbacteria and grown on nonselective media. In rare cases, the Ac-elementfailed to re-integrate or integrated into a sister chromatid after itsexcision. Abnormal shoots with an extra shooty phenotype were selectedand cultivated further for six months. From these, several normal shootsgrew. Some of these were a result of the transposable element Ac havingexcised from the genome along with the ipt gene, as determined by DNAanalysis. Some of these plants retained the other necessary markerswhich had also been included in the plasmid. This method thereforeovercomes the problems of having a constitutively expressed ipt genepresent. Unfortunately, this method requires many months of cultivationand results in only a few plants that have lost the ipt gene. Ebinuma etal. (1997) report that 6 months after infection the frequency of markerfree plants was 0.032%. Furthermore, the selection of “normal” shootsfrom abnormal regenerants was based on a variable morphologicalcriterion. The morphological selection also does not distinguish betweenplants that lost the 35S-ipt gene and chimeric plants or plants withvery low ipt expression level.

The use of inducible promoters is another means that has been used toovercome the problems associated with the constitutive overexpression ofthe ipt gene in transgenic plants. The use of a copper-induciblepromoter to regulate ipt expression led to the specific expression ofthe ipt gene in the roots, the major organ for cytokinin biosynthesis(McKenzie et al., 1998). In addition, regulated ipt expression by thetetracycline inducible system (Gatz et al. 1992) provided data about thebiological effects of cytokinins in plants and their transport throughthe vascular system (Faiss et al., 1997; Redig et al., 1996). Transgenicplants carrying the ipt gene under the control of heat shock (Medford etal., 1989) and light inducible promoters (Redig et al., 1996) have alsobeen reported. All of these systems were used to study the biologicaleffects of cytokinins and were not used for transformation.

The CKI1 (cytokinin-independent 1) gene was recently identified(Kakimoto, 1996). The gene encodes a putative receptor-like histidinekinase similar to the two-component regulators originally identified inbacteria. CKI1 likely acts in cytokinin recognition, or at an earlystage of signal transduction (Kakimoto, 1996). Cytokinin plays a centralrole in plant development. Typical cytokinin responses include rapidcell division and shoot formation. Overproduction of this gene in plantsresults in plants that exhibit typical cytokinin responses, includingrapid cell division and shoot formation in tissue culture in the absenceof exogenous cytokinin (Kakimoto, 1996). The CKI1 gene can be used as aselectable marker in a manner similar to ipt, i.e., the CKI1 gene can beput under the control of a promoter and overexpressed in transgenicplant cells thereby inducing shoot formation in the absence of exogenousplant hormones. Such shoots can be excised, thereby obtaining transgenicplants. Such shoots, obtained either from cells transformed with ipt orCKI1, cannot be made to grow normally while the cells overexpress thesetransgenes.

The knotted gene and knotted-like genes are a third group of genes whichwhen overexpressed can lead to ectopic production of adventitious shoots(Chuck et al., 1996; Lincoln et al., 1994). These can be used asselectable markers in the same manner as the ipt and CKI1 genes.

The LEAFY COTYLEDON (Lec1) gene encodes a transcription factor homologthat binds to a CCAAT box and that is required early and late in embryodevelopment and is sufficient to induce embryonic programs in vegetativecells (Lotan et al., 1999). LEC1 is an important regulator of embryodevelopment that activates the transcription of genes required for bothembryo morphogenesis and cellular differentiation. Mutations in LEC1cause the loss of identity of cotyledons. Embryonic leaves or cotyledonsof led mutants possess trichomes, epidermal hairs, which normally formonly on leaves and stems (Lotan et al., 1999). Overexpression of35S/Lec1 wild-type plants results in the formation of embryo-likestructures in cotyledons and plants (Lotan et al., 1999).

SERK (somatic embryogenesis receptor-like kinase) is a protein thatresembles a leucine-rich repeat receptor kinase protein (Schmidt et al.,1997). It was isolated from carrots and has homologs in both monocotsand dicots. It is specifically expressed during somatic embryogenesisand early zygotic embryos and it marks plant somatic cells which arecompetent to form embryos (Schmidt et al., 1997). SERK may act at anearly stage of the signal transduction during both somatic and zygoticembryogenesis to promote embryo formation.

There are two common pathways (organogenesis and somatic embryogenesis)to regenerate shoots from explants (see, e.g., Day, 1999; Dudits et al.,1995). For somatic embryogenesis, somatic embryos can be induced fromcallus in the presence of 2,4-D (2,4-dichlorophenoxyacetic acid). Afterremoval of 2,4-D, these somatic embryos can directly germinate intoseedlings. Most economically important species including all major cropsare regenerated via the somatic embryogenesis pathway.

In addition to the use of markers to identify transgenic plants, the useof promoters to control expression of the transgenes is a normal part ofsuch experiments. In most experiments, the transgenes are transcribedfrom a strong promoter, such as the 35S promoter of the cauliflowermosaic virus (CaMV). However, a more flexible gene expression system isneeded to extract greater benefits from transgenic technology. Goodinducible transcription systems are desired because transgenic plantswith inducible phenotypes are as useful as conditional mutants isolatedby traditional genetics. In this regard, several induction systems havebeen reported and successfully used (Ainley and Key, 1990; Gatz et al.,1992; Mett et al., 1993; Weinmann et al., 1994). Among these, thetetracycline-dependent expression systems are the most advanced (forreview, see Gatz, 1996).

The glucocorticoid receptor (GR) is a member of the family of animalsteroid hormone receptors. GR is not only a receptor molecule but also atranscription factor which, in the presence of a glucocorticoid,activates transcription from promoters containing glucocorticoidresponse elements (GREs) (for reviews, see Beato, 1989; Picard, 1993).It has been thought that the GR system could be a good induction systemin plants because it is simple, and glucocorticoid itself does not causeany pleiotropic effects in plants. Nevertheless, a general and efficientglucocorticoid-inducible system using GR has not previously beenconstructed for transgenic plants, although it has been demonstratedthat a system comprising GR and GREs could work in a transientexpression system with cultured plant cells (Schena et al., 1991). Onthe other hand, it has been reported that the (hormonal) regulatoryregion (or domains) of GR could regulate the function of planttranscription factors in transgenic plants (Aoyama et al., 1995; Lloydet al., 1994). Lloyd et al. (1994) showed that trichome development inArabidopsis could be successfully controlled by a chimeric proteincomprising the glucocorticoid regulatory domains and the maizetranscriptional regulator R. However, the construction of such achimeric transcription factor whose activity is tightly regulated by theglucocorticoid receptor domain is not always easy and achievable inevery case. Tight regulation appears to be critically dependent on theintramolecular structure of the chimeric protein, especially therelative position between the glucocorticoid receptor domain and thedomain whose function is to be regulated.

The regulatory region of animal steroid hormone receptors, which includea hormone-binding domain (HBD) and binding sites for HSP90, are thoughtto have repressive effects on covalently linked, neighboring domains inthe absence of their cognate ligands, and binding of the appropriateligand to an HBD results in de-repression (Picard, 1993). This mechanismwas taken advantage of by designing a transcription factor in which aconstitutively active transactivating function was regulated by theregulatory region of the rat GR in cis (Picard et al., 1988; Rusconi andYamamoto, 1987). A chimeric transcription factor comprising theDNA-binding domain of the yeast transcription factor GAL4 (Keegan etal., 1986) and the transactivating domain of the herpes viral proteinVP16 (Triezenberg et al., 1988) was chosen as a constitutively activetransactivating function. The chimeric protein GAL4-VP16 was thought toact as a strong transcription factor in all cell types because theactivation domain of VP 16 is known to interact directly with generaltranscription factors, which are thought to be evolutionarily conservedamong eukaryotes (Goodrich et al., 1993; Lin et al., 1991; Sadowski etal., 1988). It has been shown that the regulatory region of the humanestrogen receptor could regulate similar chimeric transcription factorsin yeast and animal tissue culture cells (Braselmann et al., 1993;Louvion et al., 1993). The HBD of the rat GR was added to the chimerictranscription factor and the resulting hybrid transcription factor wasdesignated ‘GVG’ because it consists of one domain each from GAL4, VP16and GR. A DNA fragment encoding the GVG transcription factor was placedbetween the cauliflower mosaic virus 35S promoter (Odell et al., 1985)and the poly(A) addition sequence of the pea ribulose bisphosphatecarboxylase small subunit rbcS-E9 (Coruzzi et al., 1984). As a bindingsite for GVG, a DNA fragment containing six copies of the GAL4 UAS(Giniger et al., 1985) was fused 5′ to the minimal CaMV 35S promoter(−46 to +9).

The publications and other materials used herein to illuminate thebackground of the invention, and in particular cases to provideadditional details respecting the practice, are incorporated herein byreference, and for convenience, are referenced by author and date in thetext and respectively grouped in the appended List of References.

SUMMARY OF THE INVENTION

Overexpression of the isopentenyl transferase gene (ipt) from theTi-plasmid of Agrobacterium tumefaciens can increase the endogenouslevel of cytokinin in transgenic plants leading to the regeneration ofshoots from transformed plant cells. When combined with a dexamethasone(DEX) or estradiol inducible system the controlled expression of the iptgene can be used to specifically select for transgenic regenerantswithout the need for an antibiotic-resistance marker. The combinedsystem allows high efficiency co-transformation with additional genesand produces transgenic plants without morphological or developmentaldefects.

The invention relates in one aspect to a method for selecting transgenicplants using a selectable marker that is under the control of achemically inducible promoter. The method involves the steps oftransforming a plant cell with a vector containing an ipt gene, CKI1gene or a gene from the knotted family, under the control of achemically inducible promoter; growing the plant cells in the absence ofplant hormone but in the presence of an inducer of the promoter; andexcising the shoots that develop. The invention further relates to amethod for selecting transgenic tobacco and transgenic lettuce plantsusing a selectable marker that is under the control of a chemicallyinducible promoter.

In another aspect, the invention relates to vectors that are useful formaking transgenic plants. The vectors are designed such that theyinclude a selectable marker that is under the control of a promoter thatis chemically inducible rather than constitutive.

The invention further relates to methods of using the above describedinducible vectors.

The invention relates in another aspect to a transgenic plant ortransgenic plant cell containing a vector with a selectable marker thatis under the control of a chemically inducible promoter. In one aspectof the invention the transgenic plants are tobacco or lettuce plants andthe transgenic plant cells are tobacco or lettuce cells.

The invention relates in another aspect to a method for selectingtransgenic plants using antibiotic and herbicide resistance genes thatare under the control of a chemically inducible promoter. Suchantibiotic and herbicide resistance genes can be regulated by thepresence or absence of inducer.

The invention relates in another aspect to a transgenic plant containinga herbicide resistance gene or an antibiotic resistance gene that isunder the control of a chemically inducible promoter. The inventionfurther relates to a transgenic tobacco plant or transgenic lettuceplant containing a herbicide resistance gene that is under the controlof a chemically inducible promoter.

The invention is also directed to a method of making plant somaticembryos by forming transgenic plant cells which can be induced tooverexpress a transgene, e.g., Lec1 or SERK, which causes the cells toproduce somatic embryos when the transgene is induced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the insert between the left andright borders in pTA7002. RB represents the right border and LBrepresents the left border. Restriction enzyme sites are shown above thedrawing. The restriction enzyme sites represented by abbreviations are:B-BamHI, H-HindIII, E-EcoRI.

FIG. 2 illustrates the insertion points into pMON721 of the luciferaseand the GVG constructs. The luciferase is inserted into the Not Irestriction site. The GVG is inserted into the multicloning site of thevector.

FIG. 3A is a scale showing the luminescence intensity from dark gray(lowest) to white (highest). Although shown as a scale of dark gray towhite, in fact the luminescence is a blue color. This scale is used forinterpreting the results of FIG. 3B.

FIG. 3B shows the stationary expression levels of the luciferaseactivity induced by different concentrations of DEX.

FIG. 3C shows the results of FIG. 3B plotted against DEX concentrations.The value obtained at 0 μM DEX (the basal, non-induction level) wasarbitrarily set as 1.

FIGS. 4A-C represent the induction of luciferase activity inArabidopsis. FIG. 4A is a color scale showing luminescence intensityfrom dark gray (lowest) to white (highest) (as in FIG. 3, theluminescence is blue, not gray as shown in the figure). FIG. 4Brepresents a transgenic plant grown in a pot for 3 weeks and thensprayed with a solution containing 0.5 mM potassium luciferin and 0.01%(w/v) Tween-20 and assayed for luciferase activity. FIG. 4C representsthe same plant as in FIG. 4B but here the plant was then sprayed with asolution containing 30 μM DEX and 0.01% (w/v) Tween-20. Twenty-fourhours later, the plant was sprayed again with the luciferin solution andassayed. For both FIGS. 4B and 4C, the luminescence from the plant wasimaged using a high-sensitivity camera system (Hamamatsu PhotonicSystems). Heterogeneity of the luminescence seen in the plant treatedwith DEX was caused by uneven absorption of luciferin.

FIGS. 5A-B represent the kinetics of the luc mRNA level induced by DEX.Transgenic tobacco plants carrying the GVG gene and the luc reportergene were first grown on agar medium for 14 days and then adapted togrowth in a hydroponic medium for 3 days. DEX treatment was started byadding DEX to the medium at a final concentration of 10 μM (timeindicated as 0). After 24 hours of treatment, DEX was then removed fromthe medium. Total RNA was prepared from 20 plants at each time indicatedand subjected to Northern blot analysis. cDNA fragments of the fireflyluciferase (FIG. 5A) and the GVG gene (FIG. 5B) were used as probes.Signals were imaged by the BAS-2000 system (Fuji Photo Films co.).Closed and open arrows indicate the time points of adding and removingDEX, respectively.

FIG. 6 shows the intensity and sustainability of induction by variousglucocorticoids. Transgenic tobacco plants carrying the GVG gene and theluc reporter gene were first grown on agar medium for 14 days and thentransferred to a fresh agar medium containing 30 μM of differentglucocorticoids for an additional 2 days. After the induction, plantswere transferred back to the agar medium without glucocorticoid (timeindicated as 0). Relative luciferase activities induced by DEX ()triamcinolone acetonide (◯), betamethasone (▪) and hydrocortisone (□)are plotted. The value obtained with no glucocorticoid (thenon-induction level) was arbitrarily set as 1.

FIG. 7 shows the local induction of luciferase expression byglucocorticoid spraying.

FIG. 8 shows dexamethasone-dependent regeneration of tobacco and lettuceshoots. Leaf discs from tobacco (upper row) and lettuce (lower row) weretransformed with the transformation cassette shown in FIG. 12. The plantmaterials were then grown for 40 days under inductive (10 μM DEX) ornon-inductive (0 μM DEX) conditions.

FIGS. 9A-F show luciferase activity in tobacco and lettuce regenerants.Luciferase activities were measured in 40 day-old regenerants grownunder inductive conditions (10 μM DEX) for expression of the ipt gene.Luciferase activity in regenerants was measured using a video imagingsystem with measurements integrated over 5 minutes and subtraction ofbackground from the images. The luciferase images were transformed from16 to 8 bit pictures and artificially colored for presentation. Thered/green overlay shows a superimposition of the bright-field andluciferase activity images to allow easy detection of luc positive andnegative regenerants. FIGS. 9A, 9C and 9E are tobacco and FIGS. 9B, 9Dand 9F are lettuce. FIGS. 9A-B show bright-field pictures, 9C-D areluciferase images and 9E-F are with a red/green overlay.

FIGS. 10A-D show Northern analysis of ipt and luc transcripts fromtobacco. The level of ipt and luc transcripts from 30 day-oldregenerants that had (+1 to +10) or did not have (−a to −g) detectableluciferase activity are shown. The regenerants were grown in thepresence of 10 μM dexamethasone. FIGS. 10A and 10C show ipt transcriptlevels and FIGS. 10B and 10D show luc transcript levels.

FIGS. 11A-C show segregation and Southern analysis of the luc gene intransgenic tobacco seedlings. Luciferase activity was measured in 44randomly selected seedlings. Thirty-three of the seedlings displayedluciferase activity and eleven of the seedlings did not displayluciferase activity (compare FIGS. 11A and 11B), demonstrating 3:1segregation of the dominant luc gene. Southern blot analysis of DNA fromthe seedlings is shown in FIG. 11C. Single bands were detected withuncut DNA (U) and after DNA was digested with Bam HI (B), Sac I (S), NcoI (N), and Xba I (X) and hybridized with radioactively labeled fragmentsof the luc gene.

FIG. 12 shows the transformation cassette for inducible expression ofthe ipt gene. The ipt gene from Agrobacterium tumefaciens was clonedunder the control of a glucocorticoid-responsive promoter (6xUAS fusedto −46 of the CaMV 35S minimal promoter) to allow regulated expressionof the gene. Expression of the ipt gene is mediated by aglucocorticoid-activated transcription factor (GVG) as described byAoyama and Chua (1997). The genes encoding hygromycin phosphotransferase(hpt) (Waldron et al., 1985) and firefly luciferase (luc) (Millar etal., 1992) were cloned under the control of constitutive promoters (NP,NOS promoter; 35S; CaMV 35S promoter) to allow easy detection oftransformation and co-transformation efficiencies. The above genes werecloned between the left and right border (LB, RB) of the T-DNA (Klee etal., 1987; Beavan and Chilton, 1982) (pBI 101, Clontech, Inc.) from theTi plasmid of Agrobacterium tumefaciens to allow Agrobacterium-mediatedtransformation.

FIG. 13 is a schematic map of the XVE vectors. The backbone of thesevectors are pTA211 and pTA221 (Kost et al., 1998), respectively. Onlyregions to be integrated into the plant genome (between the right borderor RB and the left border or LB) are shown (not to scale). G1090: asynthetic promoter (Ishige et al., 1999) driving XVE; XVE: a chimerictranscription factor containing the DNA-binding domain (DBD) of LexA(residues 1-87), transcription activation domain of VP16 (413-490) andthe regulatory region of the human estrogen receptor (272-595); E⁹T:rbcs E9 polyA addition sequence; NOS: nopaline synthase promoter; HPT:hygromycin selection marker; KAN: kanamycin selection marker; NOST:nopaline synthase poly A addition sequence; 8XLexA: 8 copies of LexArepressor binding sites; −46: the 35S minimal promoter; 3A_(T): rbcs 3ApolyA addition sequence; MCS: multicloning site.

FIGS. 14A-B show expression of the GFP gene controlled by the XVEinducible system. FIG. 14A shows roots of a pER8-GFP transgenicArabidopsis line. The GFP signals (green) emitted from the same rootswere viewed under a fluorescence microscope as shown in FIG. 14B.

FIG. 15 shows the dose-dependence on 17-β-estradiol of the XVE induciblesystem. Three-week-old pER8-GFP transgenic plants cultured in theabsence of the inducer were transferred onto medium containing variousconcentrations of 17-β-estradiol, and incubated for 16 hours. RNAs wereprepared from not-treated (lane 0; control) or 17-β-estradiol-treatedplants, and analyzed by Northern blotting using the GFP cDNA as a probe.Numbers above each lane indicate the concentrations (in micromolar) ofthe treatment.

FIG. 16 shows the induction time-course of the XVE system.Three-week-old pER8-GFP transgenic plants cultured in the absence of theinducer were transferred onto medium containing 2 μM 17-β-estradiol, andincubated for various times (indicated in hours above each lane).Analysis of the GFP transcripts was carried out as described in FIG. 15.

DETAILED DESCRIPTION OF THE INVENTION

In both basic research and practical applications, it is highlydesirable to express genes in a controllable fashion. To establish aninducible expression system, several critical factors must beconsidered. First, the system must be highly inducible. Second, thesystem must be tightly controlled and respond only to specific inducers.Third, upon induction, the system specifically activates desired geneswithout alteration of the expression of others. Lastly, the inductionitself should not have physiological or other effects on hosts. Thisdisclosure sets forth inducible expression systems for use in plants.

We reasoned that inducible systems can enable the development ofprotocols using the ipt gene as a transformation marker without thedrawbacks of constitutive expression. Under inductive conditions, cellstransformed with the ipt gene should have elevated cytokinin levels andhence the potential to regenerate shoots from plant calli or explants.In this context, the overexpression of the ipt gene can serve as anantibiotic-free marker system that specifically selects for transformedcells. As described by Aoyama and Chua (1997), the dexamethasoneinducible system allows tightly regulated expression of target genes intransgenic plants. This system consists of a hybrid transcription factorthat mediates transcription when activated with DEX and a regulated geneunder the control of cis elements that respond only to thistranscription factor. Hence, we used a transformation cassettecontaining the ipt gene under the control of the DEX-inducible systemacting as an antibiotic-free marker for the co-transformation of twoother constitutively expressed genes (hygromycin phosphotransferase(hpt) (Waldron et al, 1985) and firefly luciferase (luc) (Millar et al.,1992). This new transformation system was established for both tobaccoand lettuce using Agrobacterium-mediated transformation.

The present invention relates in one embodiment to transgenic plantsthat have been transformed with a vector that includes a selectablemarker which is under the control of an inducible promoter. In apreferred embodiment of the invention the transgenic plant is a tobaccoplant. In an alternate preferred embodiment of the invention thetransgenic plant is a lettuce plant.

In one embodiment of the invention the vector that is used to form thetransgenic plants includes a chemically inducible promoter thatactivates the selectable marker. If desired, any other gene of interestcan also be put under control of the inducible promoter such that thegene can be turned on whenever desired. Such a gene need not be amarker. Examples of such vectors are presented in the following Exampleswhich describe not only the vectors, but the methods used to prepare andscreen for transgenic plants containing such vectors.

In one embodiment of the invention the promoter can be induced in orderto select for cells or plants that have become transgenic but will notbe induced under natural growth conditions. In this manner theselectable marker gene, although present in the transgenic plants, willbe completely silent during the normal growth of the plants and shouldnot interfere with the growth of the plants. Such a silent marker genewill also be more environmentally sound than, e.g., having an antibioticresistance gene marker present wherein said resistance gene is expressedduring the normal growth of the plant. The use of this latter type ofmarker is of concern because it may lead to the development of organismsresistant to the antibiotic.

In one embodiment of the invention the inducible promoter is theglucocorticoid receptor. This has been thought to be a good inductionsystem for plants because glucocorticoid itself does not cause anypleiotropic effects in plants. In a preferred embodiment of theinvention the transcription factor that binds the glucocorticoidreceptor is a chimeric transcription factor in which the regulatoryregion of the rat GR is added to the DNA-binding domain of the yeasttranscription factor GAL4 and the transactivating domain of the herpesviral protein VP16. The resulting hybrid transcription factor isdesignated ‘GVG’ because it consists of one domain each from GAL4, VP16and GR. The GVG gene was introduced into tobacco together with aluciferase (Luc) reporter gene transcribed from a promoter containingsix copies of the GAL4 upstream activating sequence (GAL4 UAS). Goodinduction of both the luciferase activity and the luc mRNA levels wereobserved upon glucocorticoid treatment.

A major advantage of the GVG system in plants is the fact that GR andglucocorticoid, at least at the concentrations used, are nontoxic andhave no observable adverse physiological effects on plants, thusallowing the induction of target genes without pleiotropic effects. Toretain this advantage, all the other components in the GVG system werealso obtained from non-plant sources.

A further advantage of the system is that glucocorticoid possessescharacteristics that make it suitable as an inducer chemical. Becauseglucocorticoid can easily permeate plant cells, rapid gene induction canbe performed using various methods. A local induction of gene expressioncan be obtained simply by spraying with a glucocorticoid solution. It isclear that inducer chemicals accumulate in leaves to a highconcentration when whole plants are treated under open air conditions.Even under such conditions, the accumulated glucocorticoid does notcause any visible damage to leaves. The induction level can be regulatedby using different concentrations or different derivatives ofglucocorticoid. This feature can be helpful for analyzing dose-dependenteffects of induced gene products. Glucocorticoid is one of thebest-studied biological compounds and over 100 different types ofglucocorticoid derivatives are now available from commercial sources.Some of the glucocorticoid derivatives may be very stable in plantswhereas others are rapidly degraded. These types of glucocorticoid wouldbe useful for stable and transient induction, respectively. Moreover,some glucocorticoid antagonists might be used for down-regulation ofinduction.

Although specific constructs are described below, others may be easilyenvisioned and produced by one of skill in the art. The GVG systemdeveloped here is very flexible in its composition. For example, thetranscriptional induction can be limited to a specific tissue byreplacing the 35S promoter for the GVG gene with a tissue-specificpromoter. Each functional domain in the GVG fusion protein is alsoexchangeable, allowing further refinement of the system. With adifferent DNA-binding domain and the regulatory region of anothersteroid hormone receptor, it is possible to develop another steroidinduction system that can be used in combination with the GVG system.

Another construct has also been developed which has advantages over orin conjunction with the GVG system. This construct is referred to asXVE. It is similar to the GVG system but contains the DNA binding domainof the bacterial repressor LexA and the regulatory region of humanestrogen receptor. The XVE construct can be used in place of the GVGconstruct wherever the GVG construct is described throughout thisdisclosure so long as the proper inducer is used for the construct beingused. The XVE construct can be used together with the GVG construct andcan be controlled separately from the GVG construct.

In a preferred embodiment of the invention the selectable markerutilized is the ipt gene. When this gene is induced it results in theextreme shooty phenotype in which plant cells grow many shoots ratherthan roots. This phenotype is easily selected by visual inspection. Oncethe inducing agent is removed, the ipt gene becomes silent and the cellsare able to grow normally. In other embodiments of the invention otherselectable markers, e.g., the CKI1 gene may be used in a similarfashion. Again, whatever marker is used will be active only whileinduced and will be silent once the chemical inducer is removed.

A variety of DNA constructs can be made that incorporate the principleof using a chemical inducible marker. The theory behind the design ofthe plasmids, which are described in detail below, was to assembleregions within a plasmid which could be well controlled.

Another embodiment of the invention relates to a method for selectingtransgenic plants using a selectable marker that is under the control ofa chemically inducible promoter. In a preferred embodiment of theinvention the ipt gene is placed under the control of a glucocorticoidinducible promoter within a plasmid. In an alternate preferredembodiment the CKI1 gene or one of the genes of the knotted family isplaced under the control of a glucocorticoid inducible promoter within aplasmid. The dexamethasone inducible system consists of a hybridtranscription factor that mediates transcription from the glucocorticoidreceptor in the presence of DEX. This system allows tightly regulatedipt expression in transgenic plants. Plant cells are transformed withthis plasmid and the cells are grown on MS medium without plant hormonesbut in the presence or absence of dexamethasone, a syntheticglucocorticoid analog. Under inductive conditions, cells transformedwith the ipt gene will have elevated cytokinin levels and willregenerate shoots from plant calli or explants. Since the cells aregrown in the absence of plant hormones, shoots will develop only incells that are transformed and overproduce cytokinins in the presence ofdexamethasone. Nontransformed cells will not produce shoots and cellsgrown in the absence of dexamethasone will not produce shoots.Overexpression of the ipt gene can thus serve as an antibiotic-freemarker system that specifically selects for transformed cells. Thissystem could also serve as a second marker to introduce additional genesinto plants that are already resistant to antibiotics. Teratoma shootsshould appear in 2-3 weeks on transformed cells grown in the presence ofdexamethasone. These shoots can be excised and placed on MS mediumcontaining indole acetic acid but without dexamethasone. Under thiscondition, the ipt, CKI1 or knotted gene should no longer be activatedand after the cytokinin level has decreased to the normal level thetransgenic plants should appear normal and fertile and be able to setseeds.

It must be noted that although some plants behave as described above(the only shoots produced are those from transformed plants), someplants may grow shoots in hormone free medium even if they are nottransformed. A variety of techniques may be used with such plants toyield successful results of selecting transformed plants. One suchmethod is that although shoots may be produced by nontransformed plants,such shoots look normal (wild-type) whereas transformed plants have theshooty phenotype. Therefore one can use the phenotype to distinguishtransformed shoots from nontransformed shoots. An alternative method isto add a hormone such as an auxin to the growth medium to suppress shootformation from nontransformed explants. This will decrease thebackground noise level of nontransformed shoots appearing. The amount ofauxin to be added can be determined by a titration, i.e., usingdifferent concentrations of auxin, to determine the level whichsuppresses growth of shoots in nontransformed explants but allows shootgrowth in transformed explants.

In one embodiment of the invention a transformation cassette (FIG. 12)containing the ipt gene under the control of the GVG glucocorticoidinducible system (Aoyama and Chua, 1997) acting as an antibiotic-freemarker was used for the co-transformation of two other constitutivelyexpressed genes (hygromycin phosphotransferase (hpt) and fireflyluciferase (luc)). When induced with DEX, isopentenyl transferase wasexpressed from the ipt gene, leading to elevated cytokinin levels. Underinductive conditions for ipt expression, the elevated cytokinin levelsled to efficient regeneration of transgenic shoots from tobacco orlettuce explants (FIG. 8). Determination of the ipt transcript levels inthe regenerants revealed that regeneration was tightly coupled to iptexpression (FIGS. 10A-D). Even under non-inductive conditions where onlya few shoots were regenerated from the explants, at least 50% of theregenerants contained the transgene. Southern and segregation analysesof transgenic shoots and plants revealed that the majority ofregenerants contained only a single copy of the ipt gene (FIGS. 11A-C).Time course experiments demonstrated that regeneration was rapid and thespecificity of the process was maintained over a time period of at least20 days. The effects of the cytokinins were thus local and the hormonesdid not diffuse and trigger the regeneration of untransformed cells.This finding is in good agreement with the observation that even highexogenously applied concentrations of cytokinin cause more or less localreactions. The efficiency of the co-transformation of the hpt and lucgenes was determined by measuring Luc activity (FIGS. 9A-F) andanalyzing regenerants for hygromycin resistance. Northern analysis wasalso performed to determine hpt and luc transcript levels (FIGS. 10A-D).In about 80% of the shoots the luc and hpt genes were successfullyco-transformed with the ipt inducible system. After the regenerants weretransferred to non-inductive conditions the morphology of the tobaccoand lettuce plants was completely normal. More than 40% of the tobaccoregenerants developed strong root systems within 20 days and couldeasily be transferred to soil. The resulting plants showed nomorphological or developmental abnormalities and the transgenes weretransmitted to the progeny. These results demonstrate the advantagesthat inducible ipt expression has over constitutive expression of ipt.

In another embodiment of the invention, antibiotic or herbicideresistance genes are placed under the control of a glucocorticoidreceptor inducible promoter. The promoter can be induced to allow forthe expression of the antibiotic or herbicide resistance genes in orderto select for transformed plant cells. Once transformed plant cells havebeen selected, the expression of the antibiotic and herbicide resistancegenes can be repressed. This system is more environmentally sound than asystem in which the transformed plants constitutively express activeantibiotic or herbicide resistance genes.

The chemically inducible system can be used more generally and of courseis not limited to being used to induce the ipt, CKI1 or knotted gene orother selectable marker. It can be used to chemically induce any gene ofinterest. It can be used to induce a screenable marker, such asluciferase or other desired screenable marker.

The development of the system which is used took place as a series ofsteps to test the individual aspects of the final construct. These stepsare set out in the following Examples. A brief introduction explainingthe progression of the experiments is first set forth here. The GVGsystem was first used to show that a construct could be made which wouldinclude a gene inducible by DEX or a glucocorticoid analog. The plasmidpMON721 was used for this purpose with luc being placed under thecontrol of UAS. This was used to make transgenic tobacco plants whichwere selected on kanamycin medium. These experiments showed that such asystem would work (Aoyama and Chua, 1997). Next, with the desire toavoid antibiotic resistance as a marker, new constructs were designed touse the ipt gene as a marker. Constructs were made with pTA7001 orpTA7002 vectors with multicloning sites downstream of the 6XUAS. Theseconstructs included the GVG chimeric transcription system under a 35Spromoter and also included a hygromycin-resistance gene regulated by theNOS promoter. The ipt gene was placed downstream of 6XUAS. Use of thisconstruct demonstrated that the “shooty” phenotype resulting from iptoverexpression could be used as a marker. Different constructs were thenmade to extend the results to plants other than tobacco. The PTA7002/iptconstruct was modified so that the 35S promoter, which is used toexpress the GVG coding sequence, was replaced with a synthetic promotercalled G10-90 which acts as a stronger promoter than the 35S promoter.This consists of 4 copies of a G box fused to the −90 35S promoter.Furthermore, an additional gene, 35S-luc was added. This construct wasused in both tobacco and lettuce plants. Selected shoots were thentested for luciferase expression and hygromycin resistance. The resultsindicate that a very high percentage of the shooty regenerants showedboth luciferase expression and hygromycin resistance. This proves thatuse of the GVG system and the ipt gene allows one to use the shootyphenotype as a marker in different plants.

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

EXAMPLE 1 DNA Constructs

A) Construct pTA7002

Plasmid pTA7002 is similar to pBI101 (Clontech) except that the sequencebetween the right border and the left border is replaced by threetranscription units. The insert between the right and left borders ofpTA7002 is illustrated in FIG. 1 and comprises a plasmid which includesthe following elements: a 35S promoter, a GAL4 DNA binding domain, aVP16 transactivating domain, the glucocorticoid receptor regulatorydomains and a pea ribulose bisphosphate carboxylase small subunitrbcS-E9 poly(A) addition sequence all as part of a first transcriptionunit (35S-GVG-E9); a nopaline synthase (NOS) promoter, hygromycinphosphotransferase coding sequence, and the NOS terminator as part of asecond transcription unit (NOS-HPT-NOS); and 6 tandem copies of a GAL4upstream activating sequence (UAS) placed upstream of a minimal 35Spromoter (−46 to +8) including the TATA region as part of a thirdtranscription unit (6xUAS-(−46/35S)-3A). This third transcription unitalso includes restriction sites (XhoI and SpeI) for insertion of anydesired coding sequence and the pea ribulose bisphosphate carboxylasesmall subunit rbcS-3A (Fluhr et al., 1986). A coding region which isinserted in the XhoI-SpeI site should contain both the initiation andtermination codons.

In more detail, the 35S-GVG-E9 transcription unit includes bases −343 to+9 of the CaMV 35S promoter (Odell et al., 1985). The GAL4 DNA bindingdomain comprises amino acids 1-74 (Laughon and Gesteland, 1984). TheVP16 acidic domain comprises amino acids 413-490 (Dalrymple et al.,1985). The GR receptor domain comprises amino acids 519-795 (Miesfeld etal., 1986). The 3′ end of this transcription unit is the poly(A)addition sequence of the pea ribulose bisphosphate carboxylase smallsubunit rbcS-E9 (Coruzzi et al., 1984). The 35S promoter which drivesthe GVG gene can be changed to a promoter fragment of choice using theSse8387I and PmeI restriction enzyme sites. By doing so, a promoter canbe inserted which can induce the inserted gene in a specific tissue orduring a specific period depending on the characteristics of thepromoter.

B) pTA7001

This plasmid is identical with pTA7002 except for the orientation of thefragment containing 6xGAL4 UAS-TATA-cloning sites-3A terminator.Therefore it also contains both the cis- and trans-elements in the T-DNAregion of the plasmid. The trans-element is the GVG region consisting ofthe GAL4 DNA binding domain, the VP16 transactivating domain, and the GRreceptor domain driven by the 35 S promoter. The cis-element consists of6xGAL4 UAS and the TATA region of the 35 S promoter. Again, this plasmidis based upon pBI101 (Clontech) with the region between RB and LB havingbeen replaced. In pTA7001 this region has become:

1-39: pTiPOST37 from pBI101 (RB=1-25)

47-858: 35S promoter from pBI221 (TATA=813-816)

867-1097: GAL4 (aa 1-77)

1117-1340: VP16(aa413-490)

1347-2180: rat GR (aa 519-795)

2207-2764: pea rbcs-E9 terminator

2780-3112: NOS promoter from pBI101

3120-4145: hygromycin phosphotransferase

4147-4399: NOS terminator from pBI101

4893-4423: pea rbcs-3A terminator

4941-4894: cloning sites XhoI, SpeI

4995-4942: 35S promoter TATA region (TATA=4980-4977)

5197-4996: 6xGAL4 UAS

5198-5357: M13mmp19 EcoRI-HaeII fragment from pBI101

5358-5862: pTiPOST37 from pBI101 (LB=5838-5862).

C) pTA7002/ipt

This plasmid was prepared by inserting a restriction fragment (XhoI,SpeI) containing the isopentenyltransferase (ipt) gene of the pTiT37plasmid (Goldberg et al., 1984) downstream of the 6xUAS promoter in thepTA7002 plasmid.

D) pMON721/Luc

This plasmid is similar in design to the pTA7002 plasmid in that itincorporates the same GVG system. However, this is based upon thepMON721 vector (Monsanto Corp., St. Louis, Mo.) rather than the pTA7002plasmid. The GVG gene, which is transcribed from the −343 to +1 regionof the CaMV 35S promoter (Odell et al., 1985), was flanked at the 3′ endby the poly(A) addition sequence of the pea ribulose bisphosphatecarboxylase small subunit rbcS-E9 (Coruzzi et al., 1984). The DNAfragments encoding specific domains were produced by the polymerasechain reaction (PCR) using primers of appropriate sequences for in-framecloning. The GAL4 DNA binding domain comprises amino acids 1-74 (Laughonand Gesteland, 1984), the VP16 acidic domain comprises amino acids413-490 (Dalrymple et al., 1985), and the GR receptor domain comprisesamino acids 519-795 (Miesfeld et al., 1986). The GAL4 UAS DNA(5′-CGGGTGACAGCCCTCCG-3′ SEQ ID NO:1) was synthesized chemically and thecoding sequence for the luc gene (de Wet et al., 1987) was excised frompGEM-luc (Promega Co.). The Luc coding sequence was transcribed from sixcopies of GAL4 UAS placed 5′ to the −46 to +1 region of the 35S promoterand flanked at the 3′ end by the poly(A) addition sequence of the pearbcS-3A (Fluhr et al., 1986). FIG. 2 illustrates the points of insertioninto pMON721 of the GVG and luc nucleic acid constructs.

E) pTA7002G/ipt/luc

This plasmid is similar to pTA7002ipt but with two differences. The 35Spromoter of the pTA7002/ipt vector was replaced with a syntheticpromoter called G10-90. This latter promoter consists of 4 copies of a Gbox (GCCACGTGCC SEQ ID NO:2) fused to the −90 35S promoter. Also, a35S-luc gene was included to facilitate visual recognition oftransformants using a sensitive imaging system. This vector is shown inFIG. 12. See, Kunkel et al., 1999.

F) XVE Vectors

The backbone of the XVE vectors pER8 and pER10 (see FIG. 13) are pTA211and pTA221 (Kost et al., 1998), respectively. XVE: a chimerictranscription factor containing the DNA-binding domain of LexA (residues1-87), transactivating domain of VP16 (413-490) and the regulatoryregion of the human estrogen receptor (272-595). The second expressioncassette, which controls the gene of interest, was made by fusing 8copies of LexA binding sites to −46 of the 35S minimal promoter.

pER8-CKI1 (XVE-CKI1): an XhoI/SpeI DNA fragment containing the coding aswell as part of the 5′- and 3′-untranslated region of the CKI1 cDNA wasinserted into the same sites of pER8 vector downstream from the8XLexA-46 promoter. In this construct, the CKI1 gene was thus placedunder the control of the XVE inducible system, and its transcription canonly be activated by 17-β-estradiol or 4-hydroxyl tamoxifen.

pER8-Lex1 (XVE-Lec1): an XhoI/SpeI DNA fragment containing the coding aswell as part of the 5′- and 3′-untranslated region of the Lec1 cDNA wasinserted into the same sites of pER8 vector downstream from the8XLexA-46 promoter. In this construct the Lec1 gene was thus placedunder the control of the XVE inducible system, and its transcription canonly be activated by 17-β-estradiol or 4-hydroxyl tamoxifen.

pER8-SERK (XVE-SERK): a genomic DNA fragment containing the ArabidopsisSERK gene (without the SERK promoter and the transcription terminationsequences) was inserted into the same sites of pER8 vector downstreamfrom the 8XLexA-46 promoter. In this construct, the SERK gene ws thusplaced under the control of the XVE inducible system, and itstranscription can only be activated by 17-β-estradiol or 4-hydroxyltamoxifen.

EXAMPLE 2 Plants Transformed with pMON721 Based Vectors

The vector pMON721 can be used in combination with A. tumefaciens strainABI but is not used with A. tumefaciens strain LB4404. Strain ABI alonecan induce shoots on tobacco leaf discs cultivated on MS medium withouthormone and is therefore unusable for experiments in which the marker isthe growth of shoots. The pMON721-A. tumefaciens strain ABI combinationis useful for those experiments in which other markers are beingscreened, e.g., when one is selecting for antibiotic resistance. Inthese experiments the cells are grown in medium with hormones andselection is by kanamycin resistance, and they are grown both in thepresence and in the absence of the inducer, e.g., dexamethasone.

A) Transformation of Plasmid into Bacteria

Plasmids were introduced into Agrobacterium tumefaciens. Plasmidsderived from pMON721 were placed into strain ABI (Monsanto Corp., St.Louis, Mo.) by methods well known by those of skill in the art. Forexample, for pMON721/Luc, a single colony of Agrobacterium tumefaciensstrain ABI (Monsanto Corp., St. Louis, Mo.) containing pMON72 ILuc wasselected from YEB plates containing 50 mg/L kanamycin, 25 mg/Lchloramphenicol, 100 mg/L spectinomycin and 100 mg/L streptomycin. TheAgrobacterium cells were transferred to a 50-mL sterile screw cap tubecontaining 10 mL YEB liquid medium with 50 mg/L kanamycin, 25 mg/Lchloramphenicol, 100 mg/L spectinomycin and 100 mg/L streptomycin. Theculture was grown at 28° C. for 24 hours. Agrobacterium cells in culturewere collected by centrifugation at 3,000 rpm at 4° C. for 10 minutes.The cell pellet was washed once in 10 mL of YEB medium with antibioticsand then resuspended in 30 mL of B5 medium, which was used forinoculation of explants. YEB medium is prepared by bringing to 1.0 literthe following: 5.0 grams sucrose, 5.0 grams peptone, 5.0 grams beefextract, 1.0 gram yeast extract and 0.04 gram MgSO₄.7H₂O.

B) Co-cultivation with Agrobacteria

Leaf discs of Nicotiana tabacum cv SR1 were transformed and regeneratedas described by Horsch et al. (1988) and transformation of Arabidopsiswas performed according to the method of Valvekens et al. (1988).

C) Luciferase Containing Transgenic Plants

Primary transgenic plants were allowed to self-fertilize and seeds werecollected. The transgenic progeny were germinated on MS medium(Murashige and Skoog, 1962) supplemented with 3% sucrose, 0.8% agar and100 μg/mL kanamycin for selection. T₃ homozygous plants grown on thesame agar medium for 14 days after germination were used in inductionexperiments. In some experiments, plants were transferred to ahydroponic growth medium containing 1/100 concentration of MS salts andadapted to the growth conditions for 3 days before use. In all cases,plants were exposed to continuous light and a temperature of 27° C.(tobacco) or 22° C. (Arabidopsis).

EXAMPLE 3 Plants Transformed with PTA7002 or PTA7001 Based Vectors

The vectors pTA7002 and pTA7001 may be used with A. tumefaciens strainLB4404. Unlike A. tumefaciens strain ABI, the LB4404 strain does notinduce shoots and this combination of vector and bacterial strain may beused in those experiments in which the growth of shoots is the marker.The experiments described here used pTA7002/ipt. However, the vectorused may include other genes of interest which are not under the controlof the GVG system, which other genes it is desired to transform intoplants. In these experiments, plants are selected on medium withouthormones and without antibiotics, but in the presence and in the absenceof inducer (e.g., dexamethasone). Only those cells grown in the presenceof the inducer should generate shoots. These shoots are cut, placed inmedium with auxins but without the inducer. The absence of the inducerstops the transcription of the ipt gene and auxin in the medium promotesroot regeneration. These can then be tested by Northern blot analysis orfor resistance to hygromycin to determine which regenerated plants infact are transformed.

A) Transformation of Plasmid into Bacteria

Plasmids were introduced into Agrobacterium tumefaciens. Plasmidsderived from pTA7002 or pTA7001 were placed into strain LB4404 (ClontechLaboratories, Inc.) by methods well known by those of skill in the art.For example, for pTA7002/ipt, a single colony of LB4404 containingpTA7002/ipt was selected from YEB plates containing 50 mg/L kanamycinand 100 mg/L streptomycin. The Agrobacterium cells were transferred to a50-mL sterile screw cap tube containing 10 mL YEB liquid medium with 50mg/L kanamycin and 100 mg/L streptomycin. The culture was grown at 28°C. for 24 hours. Agrobacterium cells in culture were collected bycentrifugation at 3,000 rpm at 4° C. for 10 minutes. The cell pellet waswashed once in 10 mL of YEB medium with antibiotics and then resuspendedin 30 mL of B5 medium, which was used for inoculation of explants.

B) Co-cultivation with Agrobacteria

Tobacco leaves were cut into sections of 4 mm×4 mm on a wet sterilefilter paper and then transferred to sterile, deionized water. The leafsections were immersed for several minutes in the Agrobacteria solution(in B5 medium) in a petri dish. The sections were blotted dry on a pieceof sterile filter paper and then placed on MBDK plates. MBDK mediacomposition is: MS salts−4.3 g/L; B5 vitamins−112 mg/L; 2-4-D -0.5 mg/L;kinetin−0.1 mg/L; sucrose−20 g/L; phytagel−2 g/L; pH 5.7.

(C) Shoot Regeneration

After 3 days of co-cultivation of tobacco leaves with Agrobacteria, theexplants were washed 3 times by immersions in 30 mL sterile watercontaining 200 mg/L carbenicillin in a petri dish. After having blotteddry on sterile paper toweling, the explants were placed on MBC mediumwith or without dexamethasone (DEX, 30 μM). MBC media composition is: MSsalts−4.3 g/L; B5 vitamins−112 mg/L; sucrose−20.0 g/L; carbenicillin−200mg/L; phytagel−2.0 g/L; pH 5.7. The plates were incubated in a tissueculture room at 25° C. and 16 hour light/8 hour dark. After two weeks,green shoot buds appeared at wound sites of the explants only on mediumcontaining DEX (30 μM). The shoots were excised and transferred to MBCIplates. MBCI medium is: MS salts−4.3 g/L, B5 vitamins−112 mg/L,sucrose−20.0 g/L, carbenicillin−200 mg/L, phytagel−2.0 g/L, pH 5.7,indole acetic acid (IAA)−0.15 mg/L.

(D) Selection of Transgenic Plants

After 10 days of culture on MBCI plates, many adventitious shootsappear. These are cut and transferred to new MBCI plates. These shootsbecome normal looking after 10 days of culture. They regenerate rootsand grow to plantlets of 4-6 leaves after 2-3 weeks. At this stage, theyare ready to be tested to verify whether they are indeed transformed.Since the pTA7001 or pTA7002 plasmid contains a NOS-Hpt gene,transformed shoots should be resistant to hygromycin. Therefore, leafsamples containing petioles are excised and transferred to MBCI mediumwith 40 mg/L hygromycin for root induction. Only ˜10% of the shootscollected are actually transformed. Nontransgenic cells may form shootsas a result of absorbing cytokinins produced from neighboring cellswhich are transformed and are producing cytokinins. Growth of theselected shoots in the presence of hygromycin can be used to select fortransformed shoots. Northern or Southern blot analysis is another meansof testing for transformation. These latter methods are useful inexperiments in which the NOS-hpt gene has been deleted from the pTA7001or pTA7002 plasmid and a gene of interest has been inserted in itsplace. Rooted shoots are transferred to pots and grown to maturity in agreenhouse. The transgenic plants appear normal and are fertile and setseeds.

EXAMPLE 4 Induction with Glucocorticoid

All glucocorticoid derivatives, dexamethasone (DEX), triamcinoloneacetonide, betamethasone and hydrocortisone were purchased from WakoPure Chemical Industries. The chemicals were dissolved in ethanol at 30mM before use and diluted in either the growth medium or the sprayingsolution. The same volume of ethanol was added to negative controlmedium or solution. In the case of tissue culture experiments (as inExample 3) DEX is included in the tissue culture medium with phytagel.In the case of whole-plant treatment, plants were grown on an agarmedium containing glucocorticoid or their roots were submerged in ahydroponic growth medium containing glucocorticoid at 0.01 mM. For thespraying method, the solution contained 30 μM DEX and 0.01% (w/v)Tween-20; the latter was added as a wetting agent. In experimentsinvolving spraying of one half of a leaf, the other half and other partsof the plant were covered with a plastic film. It should be noted thatalthough DEX is not an especially toxic chemical, it could have somephysiological effect on a human and one should take precautions,especially the use of eye protection when one is spraying the compound.

EXAMPLE 5 Luciferase Assays

Extraction of luciferase and assays for relative luciferase activitieswere carried out as described by Millar et al. (1992). To image theluciferase luminescence, roots of plants treated with DEX were submergedin a solution containing 0.5 mM potassium luciferin (Sigma) for 1 houror the petiole of a sprayed leaf was submerged in a solution of 0.5 mMpotassium luciferin for 30 minutes. Potted plants were sprayed with asolution containing 0.5 mM potassium luciferin and 0.01% (w/v) Tween-20and left for 30 minutes. The luciferase luminescence from plants wasvisualized using an image-intensifying camera (VIM) and photon-countingimage processors (ARGUS-50) purchased from Hamamatsu Photonic Systems.The exposure time was 10 minutes. To take a picture of the luciferaseluminescence from the sprayed leaf, the leaf and an instant color film(LP100, Fuji Photo films co.) were placed in contact with one another,with a thin plastic film between them, for 5 hours.

EXAMPLE 6 RNA Analysis

Total RNA isolation and Northern blot hybridization were performed asdescribed by Nagy et al. (1988). After hybridization, signals wereimaged with the BAS-2000 system (Fuji Photo Films Co.).

EXAMPLE 7 Selection of the Best Transgenic Lines

Several independent transgenic lines should be obtained and tested. Oneshould select the best line as that which has a low basal level and ahigh induction level. Multicopies of T-DNA fragment are often insertedinto one locus. In such a case, the 35S-promoter near the RB mighthappen to neighbor the inducible promoter and change the induciblepromoter to a constitutively active promoter. Other than such a case, achromosomal sequence neighboring the inducible promoter might alsoaffect the activity. Therefore it is best to test the obtainedtransgenic lines to find one which has low basal activity and a highinduction level.

EXAMPLE 8 Induction of Luciferase Activity in Transgenic Plants

Stationary induction levels of the luciferase activity were measured inresponse to different concentrations of a glucocorticoid. Youngtransgenic plants (prepared using the pMON721/luc vector) grown on anagar medium were transferred to a fresh agar medium containing differentconcentrations of DEX. After 2 days on the induction medium, whole celllysate was prepared from 10 plants and assayed for luciferase activity.FIG. 3B shows an image of luciferase luminescence from plants using ahigh-sensitivity camera system. The color scale for FIG. 3B is shown inFIG. 3A. FIG. 3C shows the relative luciferase activity induced bydifferent concentrations of DEX. The luciferase activity detected in theabsence of DEX was very low and comparable to that obtained fromtransgenic plants carrying a luciferase gene preceded by the TATA regiononly (data not shown). This result indicates that the GAL4 UAS wasquiescent in plants and not recognized by any endogenous planttranscription factor. Induction was detectable at a concentration of 0.1μM DEX or higher, and a good correlation between DEX concentrations andinduction levels was obtained in the concentration range from 0.1 to 10μM. The maximum induction level was 100 times the basal level.

In this experiment, plants were treated with DEX for a sufficiently longperiod to ensure that the luciferase activity had reached a plateau foreach DEX concentration. Induction was very slow in plastic wares, asobserved in this experiment, probably because, under the enclosedconditions, transpirational water flow in plants and hence the uptake ofglucocorticoid through the roots was slow compared with that undernon-enclosed, open-air conditions. On the other hand, under the latterconditions, it is very difficult to precisely control the glucocorticoidconcentration in plants because the hormone rapidly accumulates inleaves, as a result of transpiration.

Various plant species have been employed for studies on basic andapplied aspects of plant sciences, and among them, Arabidopsis hasemerged as a model plant for basic explorations of plant biology. Sofar, however, good induction systems have not yet been developed forthis model plant. Induction systems using plant promoters, e.g.,heat-shock promoters, are not suitable because they elicit pleiotropiceffects. Although the tetracycline-dependent expression system has beensuccessfully used in tobacco, it does not appear to function inArabidopsis (Gatz, 1996). On the other hand, it is seen here that theGVG system can also function in Arabidopsis. FIGS. 4A-C show that theluciferase activity in transgenic Arabidopsis was induced effectively byDEX. The GVG system should be widely applicable to many genes and indifferent species of transgenic plants.

EXAMPLE 9 Kinetics of the Transcriptional Induction by DEX

Although the luciferase activity is easy to measure, it is not suitablefor kinetic study within a short time scale because the half-life ofluciferase activity is estimated to be approximately 3 hours (Thompsonet al., 1991). To obtain more direct information on the kinetics ofinduction, total RNA was prepared and subjected to Northern blotanalysis. In these experiments, plants were placed in the open air toensure rapid DEX uptake. Transgenic plants were adapted to hydroponicgrowth conditions in the open air and DEX was added to the liquid growthmedium at a final concentration of 10 μM. Total RNA was prepared from 20plants at each time point and subjected to Northern blot analysis.Results for plants transfected with pMON721/luc are shown in FIGS. 4Aand 5A. FIG. 4A shows that the luc mRNA was first detected 1 hour afterthe addition of DEX and the amount increased to a stationary levelwithin the next 3 hours. To examine the sustainability of the induction,DEX was removed from the medium and total RNA prepared from the plantswas analyzed. FIG. 5A shows that luc mRNA could be detected even 4 daysafter removal of DEX.

A similar result was obtained by monitoring the luciferase activity. Dueto the high sensitivity of detection, the induced luciferase activitycould be measured 30 minutes after DEX addition and for 8 days afterremoval of the hormone (data not shown). From these results, it can beconcluded that the transcriptional induction by DEX is rapid and can bemaintained for a long period.

EXAMPLE 10 Responses to Various Glucocorticoids

Different glucocorticoid derivatives were examined for the intensity andthe duration of induction. Young transgenic plants (transfected withpMON721/luc) grown on an agar medium were transferred to a fresh agarmedium containing 30 μM of different glucocorticoids and grown for anadditional 2 days. After the induction, plants were returned to the agarmedium without glucocorticoid. At each time point indicated in FIG. 6,10 plants were harvested and their luciferase activities assayed. FIG. 6shows that the induction levels and their durations were different withdifferent glucocorticoid derivatives. The highest induction levels wereobtained with either DEX or triamcinolone acetonide. In contrast, onlylow or moderate induction levels were detected with betamethasone orhydrocorticoid, respectively. In these experiments, it was assumed thatthe induction level obtained with each glucocorticoid had reached asteady-state level because longer induction periods did notsignificantly increase the luciferase activity (data not shown). Theinduction by DEX was maintained for a longer period compared with thatby triamcinolone acetonide, whereas both glucocorticoids conferred aboutthe same induction level at the beginning of the treatment. Although thestability of these glucocorticoids in plants is not known in theseexperiments, the induction characteristics of different glucocorticoidsmight be used to regulate the intensity and the duration of induction.

EXAMPLE 11 Local Induction of Luciferase Expression by GlucocorticoidSpraying

The right and left halves of a leaf (about 10 cm in length) on a matureplant carrying the GVG and the Luc genes (the plant was transgenic forthe pMON721/luc vector) were sprayed with a solution containing 30 μMDEX and 0.01% (w/v) Tween-20 and a control solution, respectively.Twenty-four hours after spraying, the leaf was excised and allowed totake up luciferin through the petiole. FIG. 7 shows fluorescence fromthe portion of the leaf which had been treated with DEX whereas nofluorescence is seen in the portion of the leaf treated with a controlsolution without DEX. FIG. 7 was taken by placing an instant color film(Fuji Photo Films Co. LP 100) on to the leaf, with thin plastic film inbetween them, for 5 hours.

EXAMPLE 12 The XVE Inducible Expression System

It will be a great advantage in basic and applied sciences toindependently and inducibly control the expression of multiple genes. Asa first step toward this goal, we developed a new inducible expressionsystem, designated XVE (see FIG. 13). Principally, the XVE system issimilar to that of GVG, in which the regulatory region of a nuclearreceptor confers the hormonal inducibility to the heterologous DBD fusedto the former sequence. The XVE chimera contain the DBD of the bacterialrepressor LexA (X) (Horii et al., 1981; Miki et al., 1981) and theregulatory region of human estrogen receptor (E) (Greene et al., 1986).These structural features allow XVE to have different DNA bindingspecificity and to be activated by different stimuli compared to GVG.Accordingly, eight copies of LexA binding sites were fused to the 35Sminimal promoter at−46 to drive effector genes.

To test the XVE system, we inserted a cDNA encoding the greenfluorescence protein (GFP) into the effector cassette of an XVE vector(pER8; see FIG. 13 for details). The pER8-GFP vector was transformedinto Arabidopsis and tobacco, and expression of the GFP gene wasassessed. Similar results were obtained from both species. Here, wepresent data obtained from a detailed analysis of pER8-GFP Arabidopsistransgenic lines. We initially screened 22 independent transgenic linesby visual inspection of plants germinated in the absence (control) orpresence (induced) of inducers (a mixture of 2 μM 17-β-estradiol and 1μM 4-hydroxyl tamoxifen) under a conventional fluorescence microscope.The result of this screening is summarized in Table 1. High levelinduction was observed in more than half of the lines, and arepresentative example in given in FIG. 14. Among the remaining lines,the GFP gene either expressed at a lower level (23%) or, in a few cases,did not have detectable expression (9%). A small fraction of these linesexpressed the GFP gene in a patchy pattern (14%). These data indicatedthat XVE is a highly efficient expression system. In all examined lines,no background expression (in the absence of inducers) was detected,suggesting that the system is tightly controlled.

TABLE 1 Summary of pER8-GFP Transgenic Arabidopsis Lines GFP Signal # ofLines % Strong 12  54.5 Weak 5 22.7 No Signal 2  9.2 Patchy 3 13.6 Total22  100  

EXAMPLE 13 Characterization of the XVE Inducible Expression System

In the original screen, a mixture of 17-β-estradiol and 4-hydroxyltamoxifen, two most commonly used inducers of estrogen receptors, wereused. To distinguish which compound is the active form for the response,these two chemicals were separately tested for their inducibility.Whereas both chemicals were capable of inducing the expression of thereporter gene, 4-hydroxyl tamoxifen appeared to be slightly less activethan 17-β-estradiol. The latter inducer was used in all subsequentexperiments.

To examine dose-dependence of the system, three-week-old seedlingsgerminated in the absence of the inducer were transferred to mediumcontaining various concentrations of 17-β-estradiol, and incubated for16 hours. RNAs were prepared from the not-treated (control) or treatedseedlings, and analyzed by Northern blotting using the GFP cDNA as aprobe. As shown in FIG. 15, the GFP transcript could be detected withtreatment of 0.0004 μM (0.4 nM) 17-β-estradiol, and the induction wassaturated at a concentration of approximately 1.25 μM.

In time course experiments, three-week-old seedlings were transferred tomedium containing 2 μM of 17-β-estradiol, and incubated for variouslengths of time. RNAs were prepared and analyzed as described before.The GFP transcript was detectable upon a 30-minute incubation, and theexpression reached the maximum level after a 24-hour induction (FIG.16). In separate experiments, the GFP fluorescence appeared unchangedupon five-week incubation on the induction medium, suggesting that thesystem remained constantly active.

Three independent transgenic lines were tested in experiments shown inFIGS. 15 and 16, and similar results were obtained. In both cases, a100-200-fold induction of transcripts was routinely achieved. Moreimportantly, no apparently toxic effects or physiological alterationswere observed in all the tested lines. The above analyses indicate thatXVE is an efficient and reliable inducible system.

EXAMPLE 14 Transfection of Tobacco Leaf Disks and Root Cells withXVE-CKI1

In addition to the GVG-ipt, pER8-CKI1 (XVE-CKI1) has been used totransfect tobacco leaf disks. Upon induction with 17-β-estradiol, shootsregenerated without using any externally applied plant hormones. Shootsinitiated 25-35 days after induction. Addition of IAA (0.15 mg/L) didnot increase efficiency, but rather had adverse effects on shootformation. The regeneration efficiency was dose-dependent on17-β-estradiol in the medium (tested at concentrations of 1, 5, 10, 20and 30 μM with saturation occurring at 10 μM).

The XVE-CKI1 vector was also used to transform tobacco root cells. Usingthe root cells, shoots were regenerated after induction with17-β-estradiol without using any externally applied plant hormones.2,4-D (0.5 mg/L) and kinetin (0.1 mg/L) were included when coculturingroots with Agrobacteria (2-3 days at 22° C.). Infected roots were placedon MBC medium with or without 17-β-estradiol (5 μM). The explants weretransferred to fresh MBC medium (with or without the inducer) every twoweeks. Explants grown in the absence of the inducer did produce white ordark-yellow calli after culturing for 20-30 days, but these would notform shoots. The explants grown in the presence of inducer formed greencallus. Shoots initiated 40-50 days after induction. On MBC medium 0 outof 49 white/brown calli became green and produced shoots, whereas on MBCmedium supplemented with 5 μM 17-β-estradiol, 13 out of 65 white/browncalli became green and produced shoots. This experiment used the samenumber of root explants with inducer as without inducer. Since 65 calliformed with inducer and only 49 without inducer, overexpression of CKI1may also increase the efficiency of callus formation.

EXAMPLE 15 Transfection of Arabidopsis Roots with XVE-CK1

A) Preparation of Root Material

Freshly harvested seeds are stored dry at 4° C. for two weeks beforeuse. Seeds are sterilized by placing them into a 1.5 mL Eppendorf tube(or other convenient container) with about 1 mL of sterilizationsolution (50% Clorox+0.01% Triton X-100) and agitating regularly for 10minutes. It is best not to use too many seeds (>1000 or about 50 μL) ina 1.5 mL Eppendorf tube because using too large a number of seedsresults in inefficient sterilization. The sterilization solution isremoved with a sterile pipette and the seeds are washed three times insterile distilled water using 1.0-1.5 mL for each rinse.

The sterilized seeds are suspended in about 0.5 mL of a sterile 0.15%agar in water solution and then spread onto the surface of A plates (MSsalts+30 g/L sucrose+0.8 g/L agar, pH 5.7). The seeds are vernalized at4° C. for two days to improve seed germination frequency. The seeds arethen incubated in a culture room and germinate in three days. One weekseedlings are used for root culture.

Ten to 15 seedlings are transferred to a 250 mL Erlenmeyer flaskcontaining 100 mL B5 medium (B5 salts+30 g/L sucrose+0.5 g/L MES(2-[N-morpholino]ethanesulfonic acid), pH 5.7). The flask is looselysealed with two layers of aluminum foil and placed on a shaker set at125 rpm. Cultures are illuminated with dim light at 22° C. After 10-15days in culture in B5 medium, the roots are used for transformation.White roots should be selected for transformation. Yellow or slightlybrownish roots cannot be transformed well.

B) Pretreatment of Root Explants

The following steps should be performed in a sterile hood. Rootsprepared as in step (A) are transferred to a sterile Petri dish. Asterile scalpel is used to cut off the root system from the plantlets.The roots are cut into approximately 1 cm segments and placed onto asterile paper towel to blot up excess medium. The root segments are thentransferred onto F1 plates (B5 salts+20 g/L glucose+0.5 g/L MES+0.5 mg/L2,4-D+0.05 mg/L Kinetin+2 g/L phytagel, pH 5.7 ) using sterile forceps.The roots are spread out so that they are all in contact with themedium. The plates are sealed with gas-permeable tape and are incubatedin a tissue culture room for 2-3 days.

C) Growth of Agrobacterium

Agrobacteria (strain LBA4404; Clontech) were transformed with constructspER8-CKI1 (XVE-CKI1), and the resulting transformants were cultured inYEB medium (5 g/L sucrose, 5 g/L peptone, 5 g/L beef extracts and 1 g/Lyeast extracts, 0.04 g/L MgSO₄.7H₂O, pH 7.0) supplemented with 100 mg/Lspectinomycin and 100 mg/L streptomycin overnight at 28 ° C. TheAgrobacteria were then pelleted and washed twice with YEB medium withoutantibiotics, and finally suspended in 2.0-2.5 mL YEB for infection ofArabidopsis root explants.

D) Inoculation of Root Explants with Agrobacterium and Cocultivation

The root explants prepared as in part B above are transferred to asterile Petri dish and cut into 0.5 cm segments. The root explants aretransferred to a sterile basket (e.g, a glass tube with a mesh cover onone end) which is put into a Petri dish which contains 20 mL B5 medium.2 mL of Agrobacterium solution from step C are placed into the B5medium. The basket is swirled gently for about 2 minutes to be certainthat the root explants are inoculated with Agrobacterium. Afterinoculation, the basket which contains the root explants is placed on 4layers of sterile paper towels to blot up excess liquid. Clumps of rootsegments are removed a few at a time from the basket using forceps andare placed onto F2 plates (F1 plates+20 mg/L acetosyringone) in clumpsof 5-10 root segments. The root segments are cocultivated withAgrobacteria for 2-3 days at 22° C.

E) Selection and Regeneration of Transformants

After cocultivation of root segments with Agrobacteria, the Agrobacteriaare washed away from the root explants by using sterile distilled watercontaining 200 mg/L carbenicillin. The root explants are collected in abasket which is then placed onto sterile paper towels to blot up excessliquid. The root segments are transferred to MIC medium (MS salts+10 g/Lsucrose+0.5 g/L MES+0.15 mg/L indole acetic acid (IAA)+100 mg/Lcarbenicillin+2.0 g/L phytagel, pH 5.7) with or without a chemicalinducer (5 μM 17-β-estradiol) and cultured at 22° C. for a cycle of 16hours of white light and 8 hours of dark. The MIC medium contains MSsalts, IAA and carbenicillin but does not contain the antibiotics forthe selection of transformants. It is noted that the presence of IAA isnot critical but does increase the efficiency of regeneration when usedwith Arabidopsis. IAA has a negative effect when used with tobacco.

The above is subcultured to the same medium after the first week andthen subcultured every two weeks. After about 10 days of culture, asmall dark green callus appears on the explants which are grown onmedium with the chemical inducer, but no green callus appears on theplate without the chemical inducer. After about 15 days, small shootsappear on the medium with the chemical inducer. After the shoots formsmall rosettes (3-4 leaves), they are transferred to MIC medium withoutthe chemical inducer to promote root regeneration. After rootregeneration, the plantlets are transferred to soil and grown tomaturity. When transferring the plantlets to soil, the agar mediumshould be washed clearly away from the plantlets. During the first twodays in soil the plantlets should be covered with a plastic wrap tomaintain high moisture.

F) Maturation of Transgenic Plants

After about 5-6 weeks, most siliques become yellow and dry. Seeds arecollected individually and stored at 4° C.

EXAMPLE 16 Transfection of Arabidopsis with XVE-Lec1

Overexpression of Lec1 under the control of the XVE inducible system(pER8-Lec1) leads to the formation of somatic embryos or embryo-likestructures in the cotyledons in transgenic Arabidopsis seedlings. Thissystem can be used to produce somatic embryos under plant hormone-freeconditions in the absence of any plant hormones. This differs from allcurrent tissue culture methods in which formation of somatic embryos isdependent on 2,4-D. After transfer onto medium without the inducer, thesomatic embryos will germinate into seedlings, thereby producingtransformants which do not express an antibiotic selectable marker.Conditional overexpression of Lec1 can also increase the efficiencies oftransformation and regeneration for monocots and gymnosperms. Usingprior art techniques, it has been very difficult to obtain regenerantsand/or transformants for most economically important species. Thismethod is particularly important for monocots and gymnosperms, whoseregeneration is mainly through the somatic embryogenesis pathway.

EXAMPLE 17 Transfection with XVE-SERK

The Arabidopsis SERK gene was cloned by PCR (polymerase chain reaction).The SERK gene was placed under the control of the XVE system. TheXVE-SERK construct was used to transfect tobacco and Arabidopsis. Uponinduction, somatic embryos will form under plant hormone-free conditionsin the absence of any added plant hormones. Prior art techniquesrequired the presence of 2,4-D for formation of somatic embryos. Aftertransfer to medium without the inducer, the somatic embryos willgerminate into seedlings, thereby producing transformants which do notexpress an antibiotic selectable marker. Conditional overexpression ofSERK will also increase efficiencies of transformation and regenerationfor monocots and gymnosperms, which previously have been very difficultto regenerate and/or transform. This method is particularly importantfor monocots and gymnosperms whose regeneration is mainly through thesomatic embryogenesis pathway.

EXAMPLE 18 Dual-inducible Systems

A major goal is to develop a double inducible expression system in whichmultiple genes can be independently and inducibly regulated. TransgenicArabidopsis plants carrying a) XVE and b) GVG can be generated bycotransformation or crosses between individual lines. Each component inthe dual-inducible system will operate independently and will notinterfere with one another and they will maintain their inducibility andtight control. Genes under the control of each promoter can be inducedin either order or simultaneously by proper use and timing of inducer.

EXAMPLE 19 Use of a Knotted Gene to Induce Shoot Formation

The knotted1 gene and its family members, e.g., the knotted1 homologousgenes KNAT1 and KNAT2, are highly expressed in shoots (Lincoln et al.,1994; Chuck et al., 1996). Transgenic plants which have been transformedwith, e.g., the KNAT1 gene under the control of a CaMV 35S promoter havesevere alterations including ectopic shoot formation (Lincoln et al.,1994). However, such shoots are unable to develop normally because ofthe uncontrolled expression of the knotted gene. A system in whichplants are transformed with knotted genes which are regulatable willallow one to produce plants which will produce shoots and then to usethe shoots to regenerate normal plants by shutting off the expression ofthe gene in the shoots. The present invention is one method ofaccomplishing such a result. A knotted gene, e.g., kn1 from maize, isplaced in a vector such that it is under the control of the GVG systemdescribed above. Plants which have been transformed with this vectorwill grow normally in the absence of an inducer of the GVG or XVEsystem. Explants, e.g., leaf disks, of these transgenic plants can betreated with an inducer (e.g., dexamethasone or 17-β-estradiol) tostimulate the development of adventitious shoots. The developed shootscan be excised and transferred to a medium without the inducer. Theseshoots will then develop normally to yield transgenic plants. Thevectors used may include other genes of interest, which are not underthe control of the GVG or XVE system, which it is desired to transforminto the plants. The selected plants will include the gene of interestand will have been selected without the requirement of using anantibiotic selectable marker. Note that the selection for transformedshoots should be performed as in Example 3, i.e., on medium withouthormones (MBC) but with carbenicillin to kill Agrobacteria. Homologs ofthe maize knotted gene from other monocot or dicot plants may be usedfor the same purpose.

EXAMPLE 20 Use of a CKI1 Gene to Induce Shoot Formation

The gene CKI1 was recently identified (Kakimoto, 1996). Overproductionof this gene in plants results in plants which exhibit typical cytokininresponses, including rapid cell division and shoot formation in tissueculture in the absence of exogenous cytokinin (Kakimoto, 1996). The CKI1gene can be used as a selectable marker in a manner similar to ipt. Asystem in which plants are transformed with CKI1 which is regulatablewill allow one to produce plants which will produce shoots and then touse the shoots to regenerate normal plants by shutting off theexpression of the gene in the shoots. The present invention is onemethod of accomplishing such a result. A CKI1 gene is placed in a vectorsuch that it is under the control of the GVG system described above.Plants which have been transformed with this vector will grow normallyin the absence of an inducer of the GVG system. Explants, e.g., leafdisks, of these transgenic plants can be treated with an inducer (e.g.,dexamethasone) to stimulate the development of adventitious shoots. Thedeveloped shoots can be excised and transferred to a medium without theinducer. These shoots will then develop normally to yield transgenicplants. The vectors used may include other genes of interest, which arenot under the control of the GVG system, which it is desired totransform into the plants. The selected plants will include the gene ofinterest and will have been selected without the requirement of using anantibiotic selectable marker. As in Examples 3 and 19, the selection isperformed on MBC plates for shoots which are then transferred to MBCIfor rooting.

EXAMPLE 21 Vectors with Antibiotic Resistance or Herbicide ResistanceGenes Under GVG or XVE Control

Antibiotic resistance causing genes have been widely used in vectors asselectable markers. One problem with such systems is that these genestend to be constitutively active and the transformed plants which areobtained will continue to express these genes. There have beenenvironmental and health concerns over inserting such constitutivelyexpressed genes into plants which are grown outside of a laboratorysetting (Bryant and Leather, 1992; Gressel, 1992; Flavell et al., 1992).Placing such genes under the control of the GVG or XVE system overcomesthese drawbacks. The antibiotic resistance genes will be expressed onlyduring the selection process at the time when an inducer is present inthe growth medium, but the genes will not be activated when grownoutside of the laboratory in the absence of glucocorticoid. Any desiredantibiotic resistance gene may be utilized. Appropriately modifiedpTA7001 and pTA7002 vectors can be utilized for this purpose. Theantibiotic gene of interest is cloned into, e.g., the XhoI-SpeI cloningsite. The pTA7001 or pTA7002 vectors will be modified such that the hptgene is inactivated or removed. These modified vectors may be used.Suitable vectors can be prepared by starting with, e.g., the pBI101(Clontech) vector. The region between the left and right borders of thevector is removed and replaced with the GVG or XVE system describedabove which includes, in brief, the 35S promoter, the GAL4 DNA bindingdomain, the VP16 transactivating domain, and the glucocorticoid receptordomain plus the 6xGAL4 UAS region followed by a cloning site or theregulatory region of the human estrogen receptor plus 8 copies of LexArepressor binding sites followed by a cloning site. Such vectors do notinclude an endogenous antibiotic resistance gene. Any desired antibioticgene can be inserted into the cloning site near the 6xGAL4 UAS region orthe 8XLexA region and will be under the control of the inducer. Thehygromycin phosphotransferase gene and the neomycin phosphotransferase(npt) gene are two examples of antibiotic genes which may be utilized.Ti-vectors which include a DEX or estrogen regulatable npt or hpt genecan be used to transform explants of the desired species. During thetissue culture phase, regenerated shoots will be selected in thepresence of DEX or 17-β-estradiol (which activates the appropriateantibiotic resistance gene) and in the presence of the appropriateantibiotic (kanamycin or hygromycin). Once verified, transgenic shootscan then be transferred into tissue culture medium with the antibioticbut without the chemical inducer. The resulting plants will contain theantibiotic resistance genes but these genes will not be active in theabsence of a chemical inducer.

Herbicide resistance genes can be similarly placed under GVG or XVEcontrol and used for selection of transformed plants during tissueculture phase. Such plants would not express the herbicide resistancegenes in the field. Examples of herbicide resistant genes are PAT(phosphinothricin acetyltransferase) which confers resistance to theBASTA herbicide (active ingredient phosphinothricin) (Rathore et al.,1993; Becker et al., 1992) and a mutant form of acetolactate synthasewhich is resistant to a sulfonylurea herbicide of DuPont (see, e.g.,Wiersma et al., 1989; Harms et al., 1992; Hattori et al., 1992; Hattoriet al., 1995). In theory these genes could be used not only asselectable markers in tissue culture but could also be expressed in thefield. Because of the possible dangers of spraying DEX or 17-β-estradiolone would not want to spray these onto plants in a field, but thismethod could be used if a safer compound is used as an inducer.

EXAMPLE 22 Plant Growth and Transformation

Nicotiana tabacum seeds cultivar SR1 were surface sterilized in 30%commercial bleach containing 0.02% Tween 20 for 10 minutes and washedfive times with sterile water. Plants were grown in a tissue cultureroom at 22° C. at 16 hour light and 8 hour dark cycles. Tobaccotransformation was essentially as described by Horsch et al. (1985) andKlee et al. (1987). Leaf discs were prepared from young leaves of fourweek old plants and the explants were cultured for two days on MB medium(MS salts, B5 vitamins, 20 g/L sucrose, 20 mg/L acetosyringone, 0.2%phytagel, pH 5.7). After a three-day co-cultivation with Agrobacteriumtumefaciens the leaf discs were transferred to MS medium containingdifferent DEX concentrations. After 20 to 40 days of culturing,regenerated shoots were excised from explants and cultured on MS mediumcontaining IAA to induce root regeneration.

Sterilized lettuce leaves (Lactuca sativa var. Great Lake #1 18) weregerminated and the seedlings were grown as described for tobacco.Transformation of lettuce leaf discs was performed as described byCurtis et al. (1996) with the exception that cytokinin was omitted fromthe culture media.

EXAMPLE 23 Optimization of the Conditions for the Induction of the iptGene in Transformed Cells

Tobacco leaf discs and lettuce leaf disks, obtained from transfectionwith pTA7002G/ipt/luc, were placed on media containing carbenicillin anddifferent concentrations of dexamethasone (DEX). The explants weretransferred to fresh medium every two weeks to maintain constant cultureconditions. The number of shoots that were regenerated from a givennumber of leaf discs increased dramatically as the concentration of DEXincreased (Table 2). One hundred seventy tobacco shoots and 198 lettuceshoots were regenerated from 28 explants at 10 μM DEX. In the absence ofDEX, only 7 shoots were regenerated in tobacco and 30 shoots regeneratedin lettuce from 28 explants each (FIG. 8).

TABLE 2 Tobacco ^(Lettuce) DEX (μM) 0 0.1 1 10 0 10 Total number ofregenerants 7 139  129  170  30 198  per 28 explants Number of examined7 72 72 72 28 28 regenerants Percentage of LUC⁺ 42  29 44 49 12 46regenerants

EXAMPLE 24 Measurement of Luc Activity in Tobacco and LettuceRegenerants

The transformation cassette pTA7002G/ipt/luc (FIG. 12) contained a lucgene under the control of the CaMV 35S promoter (Millar et al., 1992;Benfey and Chua, 1990). Luc activity was measured in order to estimatethe number of transgenic shoots using the video imaging system describedby Michelet and Chua (1996). Measurements were integrated over 5 minutesand the corresponding background was subtracted from the images (FIGS.9A-F). Approximately 50% of the regenerants expressed the luc gene(Table 2). Under non-inductive conditions (0 μM DEX) 42% of the tobaccoregenerants and 12% of the lettuce regenerants showed detectable Lucactivity (Table 2). This indicates that a small percentage of thetransformed cells were very sensitive towards cytokinin whose levelsmight be slightly elevated due to leaky ipt expression. Because of thehigh yield of regenerants the experiments described below were performedusing shoots from explants that were treated with 10 μM DEX.

EXAMPLE 25 Measurement of the Effect of Induction of ipt with DEX OverTime

To further characterize the transformation system, we examined theeffects of the duration of induction as well as the effects ofexogenously applied phytohormones. Time course experiments wereperformed to determine whether the specificity of the induction of theipt gene decreases over time due to overproduction and diffusion ofcytokinin, which could trigger regeneration events in neighboring,non-transformed cells. The Luc activity in 54 tobacco regenerants(obtained from a transfection with pTA7002G/ipt/luc) was measured after20, 30, and 40 days of induction with 10 μM DEX to estimate thetransformation frequency. No significant difference in the percentage ofregenerants with detectable Luc activity was found over time. After 20days, 46% of regenerants had detectable Luc activity and after both 30and 40 days 53% of regenerants had detectable Luc activity. In addition,there were no detectable changes in the transformation efficiency(determined as Luc activity) when ipt gene expression was directlyinduced during the co-cultivation period with Agrobacterium. Similarresults have been obtained with lettuce.

EXAMPLE 26 Influence of Exogenously Applied Auxin During Induction

Luciferase activity was measured in shoots from tobacco explants(obtained from a transfection with pTA7002G/ipt/luc) that were culturedfor 40 days on medium containing 1.0, 1.5, and 2.0 mg/mL auxin and 10 μMDEX. High auxin to cytokinin levels favored root regeneration and had asuppressing effect on shoot regeneration, and therefore, might reducethe number of non-transgenic regenerants. No significant difference inluciferase activity occurred at different auxin concentrations. At 1.0mg/mL auxin 45% of regenerants had detectable Luc activity, at 1.5 mg/mLauxin 58% of regenerants had detectable Luc activity, and at 2.0 mg/mLauxin 47% of regenerants had detectable Luc activity.

EXAMPLE 27 Determination of ipt Transcript Levels in Shoots UsingNorthern Analysis

The level of ipt transcripts was determined in 30 day-old tobacco andlettuce regenerants (from a transfection with pTA7002G/ipt/luc) with andwithout detectable luciferase activity. The regenerants were grown inthe presence of 10 μM dexamethasone.

RNA was extracted from 0.1 g of plant material using Qiagen RNAextraction kits and protocols. Total RNA was separated on 1% agarosegels containing 0.8 M formaldehyde. RNA was transferred to Duralon UVmembranes according to the manufacturer's instructions (Stratagene).After blotting, the RNA was covalently cross-linked to the membrane byUV irradiation. The membranes were blocked and hybridization wasperformed using Stratagene QuikHyb® solution at 68° C. according to themanufacturer's instructions. After hybridization the membranes werewashed three times for 15 minutes with 2×SCC+0.1% SDS at 65° C. and oncewith 0.1×SSC+0.1% SDS at 60° C. for 15 minutes.

The results of Northern analysis revealed that with only one exception(FIG. 10C, sample f) all tested tobacco and lettuce regenerantsexpressed ipt transcripts (Table 3). The amount of ipt transcript washigher in tobacco than in lettuce regenerants and varied betweendifferent shoots. Transcript levels for ipt were higher in LUC⁺ shootsthan in regenerants without detectable luciferase activity (LUC⁻). Thisindicates that regeneration was almost totally coupled to ipt geneexpression leading to elevated cytokinin levels.

TABLE 3 Number of tobacco Luciferase luc (copy regenerants activity ipttranscript luc transcript number) 9 + + + 1 1 + + + >1   3 − + shorter 11 − + shorter >1   3 − + − 0 1 − − − 0

EXAMPLE 28 Determination of the Presence of the luc Gene and the Levelof Its Transcript in Shoots Using Southern and Northern Analysis

To assay the presence of the luc gene, Southern blot analysis wasperformed using DNA from LUC⁺ and LUC⁻ tobacco regenerants (obtainedfrom transfection with pTA7002G/ipt/luc). DNA was extracted from 0.1 gtobacco plant material using Nucleon DNA extraction kits and protocols.DNA was separated on 0.8% agarose gels. DNA was transferred to DuralonUV membranes according to the manufacturer's instructions (Stratagene).After blotting, the DNA was covalently cross-linked to the membrane byUV irradiation. The membranes were blocked and hybridized usingStratagene QuikHyb® solution at 68° C. according to the manufacturer'sinstructions. After hybridization the membranes were washed three timesfor 15 minutes with 2×SSC+0.1% SDS at 65° C. and once with 0.1×SSC+0.1%SDS at 65° C. for 15 minutes. The luc gene was found in all LUC⁺regenerants and in 50% of the LUC⁻ shoots.

Northern analysis was performed according to Example 27 in order toassay the presence of luc transcripts in LUC⁺ and LUC⁻ tobaccoregenerants. LUC⁻ regenerants possessed smaller, less abundant luctranscripts compared to the luc transcripts from LUC⁺ regenerants, whichwere larger and more abundant (Table 3).

EXAMPLE 29 Analysis of Hygromycin Resistance

Expression of the hpt gene confers hygromycin resistance. To assay thepresence of the hpt gene in LUC⁺ and LUC⁻ tobacco regenerants (obtainedby transfection with pTA7002G/ipt/luc), hygromycin resistance wasassayed as discussed in Example 3. Young leaf blades with petiole wereplaced into an agar medium with hygromycin and scored for rootformation. Hygromycin resistance was tested on plates with non-inductivemedium containing 20 mg/L hygromycin. Ninety-five percent of all testedLUC⁺ tobacco regenerants were resistant to hygromycin and more than 60%of the tested LUC⁻ shoots were hygromycin-resistant.

EXAMPLE 30 Root Regeneration, Plant Morphology, and Copy Number inTobacco

Tobacco shoots (obtained from transfection with pTA7002G/ipt/luc) weretransferred to a root-inducing medium (1×MS salts, B5 vitamins, 0.15mg/L IAA, 20 g/L sucrose, 0.2% phytagel, pH 5.7) that did not containDEX. Over 40% of the transgenic tobacco shoots developed a strong rootsystem within 20 days after transfer. With very few exceptions (lessthan 2%) the morphology of the transgenic tobacco plants appearednormal. The tobacco plants were then transferred to soil. The plantsdeveloped normal leaves and flowers and were apparently unaffected inseed production.

Segregation analysis for the luc gene family was performed with 44randomly selected seedlings (T1 progeny) of one transgenic tobacco line(FIGS. 11 A-B). Seeds were taken from transgenic plants that had beenself-crossed. A population of the progeny (germinated plantlets) wereanalyzed. Luciferase activity measurements in these seedlings showed aclear 3:1 segregation of the dominant luc gene. This showed an insertioninto one locus and that the transgene was stably transmitted into thesecond generation.

Southern analysis was performed with DNA from the seedlings afterdigestion of the DNA with restriction enzymes. Single gel bands weredetected (FIG. 11C).

EXAMPLE 31 Southern Blot Analysis of Tobacco Regenerants

Southern analysis was performed with DNA from eighteen tobaccoregenerants (obtained from transfection with pTA7002G/ipt/luc) afterdigestion with Bam HI, Sac I, and Xba I. Most of the shoots containedonly a single copy of the transgene. Only two out of eighteenregenerants showed the presence of more than one hybridizing band (Table3).

While the invention has been disclosed herein by reference to thedetails of preferred embodiments of the invention, it is to beunderstood that the disclosure is intended in an illustrative ratherthan in a limiting sense, as it is contemplated that modifications willreadily occur to those skilled in the art, within the spirit of theinvention and the scope of the appended claims.

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2 1 17 DNA Saccharomyces cerevisiae 1 cgggtgacag ccctccg 17 2 10 DNAArtificial Sequence Description of Artificial SequenceThis is a G-boxsequence. 2 gccacgtgcc 10

What is claimed is:
 1. A method for selecting a transgenic plantencoding a silent selectable marker wherein said method comprises thesteps of: a) transforming a plant cell with a vector wherein said vectorcomprises DNA encoding a regulatory region of an estrogen receptor, aDNA-binding domain and a transactivating domain, and further whereinsaid vector comprises nucleic acid encoding a silent selectable markerwhich promotes shoot formation, wherein said nucleic acid is under thecontrol of an inducible promoter to which the DNA-binding domain bindsin response to an inducer which is estrogen or an estrogen derivative;b) growing said plant cell in the absence of an exogenous plant hormonebut in the presence of said inducer of said inducible promoter; c)excising shoots which develop, and d) growing said shoots to produceplants; wherein a plant produced from step (d) is selected as being atransgenic plant.
 2. The method of claim 1 wherein said gene whichpromotes shoot formation is selected from the group consisting of an iptgene, a CKI1 gene or a gene from the knotted family.
 3. The method ofclaim 1 wherein said vector further comprises one or more genes ofinterest.
 4. The method of claim 1 wherein said vector further comprisesan antibiotic resistance gene.
 5. The method of claim 1 wherein saidvector further comprises a herbicide resistance gene.
 6. The method ofclaim 1 which further comprises the step of: (e) cultivating at leastone of said shoots in the absence of said inducer to produce atransgenic plant.
 7. The method of claim 4 wherein said shoots arefurther selected by growing said shoots in the presence of anantibiotic, said antibiotic being an antibiotic to which said antibioticresistance gene confers resistance.
 8. The method of claim 5 whereinsaid shoots are further selected by growing said shoots in the presenceof a herbicide, said herbicide being a herbicide to which said herbicideresistance gene confers resistance.
 9. The method of claim 1 whereinsaid vector further comprises DNA encoding a DNA-binding domain of thebacterial repressor LexA.
 10. The method of claim 1 wherein said induceris selected from 17-β-estradiol or 4-hydroxyl tamoxifen.
 11. The methodof claim 1 wherein said vector further comprises DNA encoding a chimericprotein comprising a regulatory domain of a glucocorticoid receptor, asecond DNA-binding domain and a transactivating domain, and furtherwherein said vector comprises nucleic acid encoding a gene of interestunder the control of a second inducible promoter to which the secondDNA-binding domain binds in response to an inducer which isglucocorticoid or a glucocorticoid derivative.
 12. The method of claim11 wherein said plant cell is grown in the presence of said inducer ofsaid second inducible promoter.
 13. The method of claim 12 wherein saidsecond inducer is selected from the group consisting of dexamethasone,triamcinolone acetonide, betamethasone and hydrocortisone.